Modern medical diagnostic tests rely upon the isolation and identification of nucleic acid sequences. Nucleic acids are the components of the genetic material of organisms and are arranged sequentially in long strands to form molecules such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). For example, in order to determine whether a patient is suffering from an illness associated with a particular virus, tests have been developed to identify one or more nucleic acid sequences unique to that virus. These tests rely on the fact that probes complementary to the target nucleic acid sequence will selectively bind or hybridize to the target sequence. The probe is chemically labelled so that it can be detected by one or more analytical methods, such as fluorometry. If a sample contains the target nucleic acid sequence, the probe will bind to the target and the presence of the bound probe can be used to confirm the presence of the target sequence.
Because the target molecule may be present in extremely small quantities in many test samples, processes have been developed to increase or "amplify" the number of nucleic acid copies present so that the target sequence can be more readily identified. Several methods of target amplification chemistry have been developed, including self-sustaining sequence replication ("3SR"), polymerase chain reaction ("PCR"), and the transcriptional amplification system ("TAS"), and several variations thereof. Basically, such methods produce copies of the nucleic acid in multiple reiterative steps such that the copies produce further copies in an exponential fashion. In this way, small amounts of nucleic acids can be rapidly amplified by a factor of more than one million, thereby producing sufficient quantities of target needed for detection in diagnostic tests. For a general discussion of amplification techniques, the reader is referred to U.S. Pat. No. 4,683,202 (Mullis), which is incorporated herein by reference and made a part hereof.
Although such target amplification procedures are extremely powerful, several drawbacks have arisen with their use. The most significant problem is so-called sample "cross-contamination," that is, the contamination of one sample with amplified target from previous amplification procedures. Reagents used in current amplification methods can also be contaminated in this manner. Because of the power and specificity of amplification techniques, even extremely small amounts of nucleic acid carried in aerosols created during normal laboratory procedures or present in trace amounts on laboratory equipment can contaminate a sample and result in false positive results, seriously jeopardizing the reliability of diagnostic tests. In extreme situations, contamination problems have become so severe and widespread that it has been necessary to relocate entire laboratories.
Laboratories have developed various methods to help minimize cross-contamination. One approach is to store separate, small aliquots of reagents and to dispense these reagents using so-called "positive displacement" pipettes, so that the reagents and sample do not contact the outside environment before or during the amplification reaction. Other approaches include physically separating amplification reactions from other laboratory processing steps and/or pre-treating reagents with ultraviolet light to destroy nucleic acid fragments present in the reagents as a result of contamination.
These techniques for preventing sample and reagent cross-contamination suffer from a number of drawbacks. Physical separation makes it more difficult to perform diagnostic procedures in a single instrument, since amplification must be confined to a separate area--or even a separate room--in the laboratory. Likewise, pretreatment with UV light does not guarantee that there will be no cross-contamination, since the reagents may potentially be exposed to contamination after such pretreatment but prior to amplification.
Chemical contamination control techniques are also known. Such techniques include so-called "pretreatment," in which DNA sequences are chemically modified during amplification. As a result, DNA copies made by amplification are chemically unique from the target. If these unique copies have contaminated a sample, they can be selectively destroyed prior to each new test without destroying the target. "Post-treatment" techniques are also known, in which the DNA strands are cross-linked after amplification, making them chemically incapable of being amplified.
Such chemical contamination control techniques also have drawbacks associated with their use. The principal drawback is that such techniques tend to reduce the sensitivity of detection, either by reducing the number of amplified copies or by rendering the amplified copies more difficult to detect with probes. Thus, the existence of these techniques does not eliminate the need for physical isolation of the amplification reaction, particularly when high sensitivity of detection is desired.
Containers for physically isolating analytical samples are also known. For example, U.S. Pat. No. 2,961,228 to Moore discloses a crucible to prevent a sample from exposure to the atmosphere prior to testing. However, this device is not adapted for use in modern diagnostic procedures and, in particular, procedures which involve nucleic acid amplification.
Accordingly, a need exists for an analytical sample container which can maintain the processes of target nucleic acid amplification (including sample lysing, addition of amplification reagents, and amplification) in substantial isolation from the outside environment, thereby helping to reduce sample and reagent cross-contamination. Further, a need exists for a sample container which permits the required amplification reagents to be added to the sample while still maintaining the sample in isolation from the outside environment. A need also exists for a container which may be used in a single, automated or semi-automated instrument, but which is relatively simple in design and easy to manufacture.